Time-lapse microscopy is a powerful tool for monitoring phenotypic change, be it cell division, neurite outgrowth, cell migration, or apoptosis. The level of detail and spatio-temporal context this method provides affords significant advantage over most reductionist endpoint techniques. This is particularly true for cellular events that unfold over many hours, days, or weeks. However, capturing and analyzing images over long time periods is technically challenging, especially at a scale to support higher throughput and/or industrialized workflows.
High-content screening (HCS) approaches in microplates—where cells are typically treated, fixed, labeled, and then imaged—have partially alleviated the throughput problem, but largely at the expense of the important kinetic component. Moreover, many cell-labeling techniques can have detrimental effects on cell morphology and function, which can further compromise analyses. To date, little attention has been paid to analyzing label-free phase contrast and/or brightfield images using HCS platforms.
To address these limitations, Essen BioScience has developed a nonperturbing, fully kinetic and quantitative method called Live Content Imaging. This approach, based around the IncuCyte ZOOM™’s “live cell imaging in your incubator” platform, integrates three things: 1) The multiplexed capabilities, image-analysis tools, and quantitative metrics of HCS platforms, 2) The information content of noninvasive time-lapse microscopy, and 3) The versatility, simplicity, and ease-of-use of microplate reader systems.
The IncuCyte ZOOM captures phase and fluorescence (green, red) images from living cells. Simple to use, integrated software modules quantify images on the fly, building real-time, plate-view metrics as the assay time course and biology unfolds. Up to six microplates (96-, 384-well) are housed within a single system, providing sufficient throughput for testing small compound or gene interference libraries.
This approach is widely applicable to adherent cells. The range of exemplified kinetic phenotypic assays includes mono- and co-culture models of cell proliferation, migration, invasion, angiogenesis, neurite outgrowth, apoptosis, and cell death. Measurements of stem cell differentiation, T-cell killing, autophagy, and megakaryocyte production have also been reported.
Some of these measurements are fluorescence-based, and therefore require the use of specialized nonperturbing fluorescent probes suitable for long-term live-cell imaging, such as NucLight™ GFP/RFP. Many, however, are accessible simply through the analysis of phase contrast images. Such are the focus of this article.